Molecular and Cellular Biochemistryhttp://hdl.handle.net/2022/14015
Tue, 31 Mar 2015 21:00:34 GMT2015-03-31T21:00:34ZInverted Alu dsRNA structures do not affect localization but can alter translation efficiency of human mRNAs independent of RNA editinghttp://hdl.handle.net/2022/19125
Inverted Alu dsRNA structures do not affect localization but can alter translation efficiency of human mRNAs independent of RNA editing
Capshew, C.R.; Dusenbury, K.L.; Hundley, H.A.
With over one million copies, Alu elements are the most abundant repetitive elements in the human genome. When transcribed, interaction between two Alus that are in opposite orientation gives rise to double-stranded RNA (dsRNA). Although the presence of dsRNA in the cell was previously thought to only occur during viral infection, it is now known that cells express many endogenous small dsRNAs, such as short interfering RNA (siRNAs) and microRNA (miRNAs), which regulate gene expression. It is possible that long dsRNA structures formed from Alu elements influence gene expression. Here, we report that human mRNAs containing inverted Alu elements are present in the mammalian cytoplasm. The presence of these long intramolecular dsRNA structures within 3′-UTRs decreases translational efficiency, and although the structures undergo extensive editing in vivo, the effects on translation are independent of the presence of inosine. As inverted Alus are predicted to reside in >5 of human protein-coding genes, these intramolecular dsRNA structures are important regulators of gene expression.
Sun, 01 Jan 2012 00:00:00 GMThttp://hdl.handle.net/2022/191252012-01-01T00:00:00ZThe coronavirus endoribonuclease Nsp15 interacts with retinoblastoma tumor suppressor proteinhttp://hdl.handle.net/2022/19124
The coronavirus endoribonuclease Nsp15 interacts with retinoblastoma tumor suppressor protein
Bhardwaj, K.; Liu, P.; Leibowitz, J.L.; Kao, C.C.
Coronaviruses encode an endoribonuclease, Nsp15, which has a poorly defined role in infection. Sequence analysis revealed a retinoblastoma protein-binding motif (LXCXE/D) in the majority of the Nsp15 of the severe acute respiratory syndrome coronavirus (SARS-CoV) and its orthologs in the alpha and beta coronaviruses. The endoribonuclease activity of the SARS-CoV Nsp15 (sNsp15) was stimulated by retinoblastoma protein (pRb) in vitro, and the two proteins can be coimmunoprecipitated from cellular extracts. Mutations in the pRb-binding motif rendered sNsp15 to be differentially modified by ubiquitin in cells, and cytotoxicity was observed upon its expression. Expression of the sNsp15 in cells resulted in an increased abundance of pRb in the cytoplasm, decreased overall levels of pRb, an increased proportion of cells in the S phase of the cell cycle, and an enhanced expression from a promoter normally repressed by pRb. The endoribonuclease activity of the mouse hepatitis virus (MHV) A59 Nsp15 was also increased by pRb in vitro, and an MHV with mutations in the LXCXE/D-motif, named vLC, exhibited a smaller plaque diameter and reduced the virus titer by ~1 log. Overexpression of pRb delayed the viral protein production by wild-type MHV but not by vLC. This study reveals that pRb and its interaction with Nsp15 can affect coronavirus infection and adds coronaviruses to a small but growing family of RNA viruses that encode a protein to interact with pRb.
Sun, 01 Jan 2012 00:00:00 GMThttp://hdl.handle.net/2022/191242012-01-01T00:00:00ZLL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAshttp://hdl.handle.net/2022/14336
LL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAs
Kao, C. Cheng; San Mateo, Lani; Dragnea, Bogdan; Wu, Linda H.; Jordan, Jarrett L.; Wen, Yahong; Ranjith-Kumar, C. T.; Bhardwaj, Kanchan; Adhikarakunnathu, Sreedevi; Lai, Yvonne
Background: Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti- microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3.
Methodology/Principal Findings: Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands.
Conclusions/Significance: LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.
Sat, 01 Oct 2011 00:00:00 GMThttp://hdl.handle.net/2022/143362011-10-01T00:00:00ZViral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3http://hdl.handle.net/2022/14335
Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3
Kao, C. Cheng; Ranjith-Kumar, C. T.; Mukhopadhyay, Suchetana; Zlotnick, Adam; Valverde, Rodrigo A.; Tragesser, Brady J.; Bhardwaj, Kanchan; Chen, Alice; Yi, Guanghui; Lai, Yvonne
Toll-like Receptor 3 (TLR3) detects double-stranded (ds) RNAs to activate innate immune responses. While poly(I:C) is an excellent agonist for TLR3 in several cell lines and in human peripheral blood mononuclear cells, viral dsRNAs tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow TLR3 to respond to viral dsRNAs. TLR3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. Recombinant 1b hepatitis C virus polymerase was found to enhance TLR3 signaling in the lung epithelial BEAS-2B cells when added to the media along with either poly(I:C) or viral dsRNAs. The polymerase from the genotype 2a JFH-1 HCV was a poor enhancer of TLR3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed RNA. The 1b polymerase also co-localizes with TLR3 in endosomes. RNA-binding capsid proteins (CPs) from two positive-strand RNA viruses and the hepadenavirus hepatitis B virus (HBV) were also potent enhancers of TLR3 signaling by poly(I:C) or viral dsRNAs. A truncated version of the HBV CP that lacked an arginine-rich RNA-binding domain was unable to enhance TLR3 signaling. These results demonstrate that several viral RNA-binding proteins can enhance the dsRNA-dependent innate immune response initiated by TLR3.
Sat, 01 Oct 2011 00:00:00 GMThttp://hdl.handle.net/2022/143352011-10-01T00:00:00Z